Inhibitors of DNA methyltransferase isoforms

ABSTRACT

This invention relates to the inhibition of DNA MeTase expression and enzymatic activity. The invention provides methods and agents for inhibiting specific DNA MeTase isoforms by inhibiting expression at the nucleic acid level or enzymatic activity at the protein level.

BACKGROUND OF THE INVENTION

[0001] 1. Field of the Invention

[0002] This invention relates to the fields molecular biology, cellbiology and cancer therapeutics.

[0003] 2. Summary of the Related Art

[0004] In mammals, modification of the 5′ position of cytosine bymethylation is the only known naturally occurring covalent modificationof the genome. DNA methylation patterns correlate inversely with geneexpression (Yeivin, A., and Razin, A. (1993) EXS 64:523). Therefore, DNAmethylation has been suggested to be an epigenetic determinant of geneexpression. DNA methylation is also correlated with several othercellular processes including chromatin structure (Keshet, I., et al.,(1986) Cell 44:535-543; and Kass, S. U., et al., (1997) Curr. Biol.,7:157-165), genomic imprinting (Barlow, D. P. (1993) Science, 260:309-310; and Li. E., et al., (1993) Nature 366:362-365), somaticX-chromosome inactivation in females (6), and timing of DNA replication(Shemer, R., et al. (1996) Proc. Natl. Acad. Sci. USA 93:6371-6376).

[0005] Selig et al. discloses that the DNA 5-cytosine methyltransferase(DNA MeTase) enzymes catalyze the transfer of a methyl group fromS-adenosyl methionine to the 5 position of cytosine residing in thedinucleotide sequence CpG (Selig, S., et al.,. (1988) EMBO J.,7:419-426). To date, three DNA MeTases have been identified in somatictissues of vertebrates. Adams et al. teaches that DNMT1 is the mostabundant DNA MeTase in mammalian cells (Adams, R. L., et al., (1979)Biochem. Biophys. Acta 561:345-357). Glickman et al. teaches that DNMT1preferentially methylates hemimethylated DNA as its substrate and,therefore, it is believed to be primarily responsible for maintainingmethylation patterns established in development (Glickman, F. J., etal., (1997) Biochem. Biophys. Res. Comm. 230:280-284). Okano et al.suggest that the recently identified DNA MeTase enzymes, DNMT3a andDNMT3b, encode the long sought de novo methylation activitiesresponsible for methylating previously unmethylated DNA, to generate newpatterns of DNA methylation (Okano, M., et al., (1998) Nat. Genet.19:219-20).

[0006] DNA methylation patterns are highly plastic throughoutdevelopment and involve both global demethylation and de novomethylation events (for review, see Razin, A., and Cedar, H. (1993) EXS64:343-57). Genetic experiments have demonstrated that proper regulationof DNA methylation is essential for normal mammalian development. Li etal. disclose that mice homozygous for the targeted disruption of DNMT1(DNMT1⁻/⁻ mice) fail to maintain established DNA methylation patternsand do not survive past mid gestation (Li, E., et al., (1992) Cell69:915-926), and similarly Okano et al. disclose that the DNMT 3b⁻/⁻genotype produces embryo lethality in mice, whereas DNMT3a⁻/⁻ micedevelop to term but become runted and die at approximately 4 weeks ofage (Okano, M., et al., (1999) Cell 99:247-57).

[0007] In addition to the role DNA methylation plays in development, itis also implicated in tumorigenesis (for review, see Jones, P. A., andLaird, P. W. (1999) Nat. Genet. 21:163-167). Baylin et al. disclose thatabnormal methylation patterns are observed in malignant cells, and thesepatterns may contribute to tumorigenesis by improper silencing of tumorsuppressor genes or growth-regulatory genes (Baylin, S. B., et al.,(1998) Adv. Cancer Res. 72:141-196). Szyf et al., U.S. Pat. No.5,919,772 discloses that tumorigenicity can be reversed by reducing theexpression of DNMT1. Elevated levels of DNMT3a and DNMT3b mRNA are alsofound in human tumors, raising a question whether they may have a rolein tumorigenesis (Li, E., et al., (1992) Cell 69:915-926, Robertson, K.D., et al. (1999) Nucleic Acids Res. 27:2291-2298, and Robertson, K. D.,et al., (2000) Nucleic Acids Res. 28:2108-2113).

[0008] Therefore, there remains a need to develop agents for inhibitingspecific DNA MeTase isoforms. There is also a need for the developmentof methods for using these agents to identify and inhibit specific DNAMeTase isoforms involved in tumorigenesis.

BRIEF SUMMARY OF THE INVENTION

[0009] The invention provides methods and agents for inhibiting specificDNA methyltransferase (DNA MeTase) isoforms by inhibiting expression atthe nucleic acid level or enzymatic activity at the protein level. Theinvention allows the identification of and specific inhibition ofspecific DNA MeTase isoforms involved in tumorigenesis and thus providesa treatment for cancer. The invention further allows identification ofand specific inhibition of specific DNA MeTase isoforms involved in cellproliferation and/or differentiation and thus provides a treatment forcell proliferative and/or differentiation disorders.

[0010] The inventors have discovered new agents that inhibit specificDNA MeTase isoforms. Accordingly, in a first aspect, the inventionprovides agents that inhibit one or more specific DNA MeTase isoformsbut less than all DNA MeTase isoforms. Such specific DNA MeTase isoformsinclude without limitation, DNMT-1, DNMT3a and DNMT3b. Non-limitingexamples of the new agents include antisense oligonucleotides (oligos)and small molecule inhibitors specific for one or more DNA MeTaseisoforms but less than all DNA MeTase isoforms.

[0011] The present inventors have surprisingly discovered that specificinhibition of DNMT3a and DNMT3b reverses the tumorigenic state of atransformed cell. The inventors have also surprisingly discovered thatthe inhibition of the DNMT3a and DNMT3b isoforms dramatically inducesgrowth arrest and apoptosis in cancerous cells. Thus, in certainembodiments of this aspect of the invention, the DNA MeTase isoform thatis inhibited is DNMT3a and/or DNMT3b. In certain preferred embodiments,the agent that inhibits the specific DNA MeTase isoform is anoligonucleotide that inhibits expression of a nucleic acid moleculeencoding that DNA MeTase isoform. The nucleic acid molecule may begenomic DNA (e.g., a gene), cDNA, or RNA. In some embodiments, theoligonucleotide inhibits transcription of mRNA encoding the DNA MeTaseisoform. In other embodiments, the oligonucleotide inhibits translationof the DNA MeTase isoform. In certain embodiments the oligonucleotidecauses the degradation of the nucleic acid molecule. Particularlypreferred embodiments include antisense oligonucleotides directed toDNMT1, DNMT3a, or DNMT3b. In yet other embodiments of the first aspect,the agent that inhibits a specific DNA MeTase isoform is a smallmolecule inhibitor that inhibits the activity of one or more specificDNA MeTase isoforms but less than all DNA MeTase isoforms.

[0012] In a second aspect, the invention provides a method forinhibiting one or more, but less than all, DNA MeTase isoforms in acell, comprising contacting the cell with an agent of the first aspectof the invention. In other preferred embodiments, the agent is anantisense oligonucleotide. In certain preferred embodiments, the agentis a small molecule inhibitor. In certain preferred embodiments of thesecond aspect of the invention, cell proliferation is inhibited in thecontacted cell. In preferred embodiments, the cell is a neoplastic cellwhich may be in an animal, including a human, and which may be in aneoplastic growth. In certain preferred embodiments, the method of thesecond aspect of the invention further comprises contacting the cellwith a DNA MeTase small molecule inhibitor that interacts with andreduces the enzymatic activity of one or more specific DNA MeTaseisoforms. In still yet other preferred embodiments of the second aspectof the invention, the method comprises an agent of the first aspect ofthe invention which is a combination of one or more antisenseoligonucleotides and/or one or more small molecule inhibitors of thefirst aspect of the invention. In certain preferred embodiments, the DNAMeTase isoform is DNMT1, DNMT3a, or DNMT3b. In other certain preferredembodiments, the DNA MeTase isoform is DNMT3a and/or DNMT3b. In someembodiments, the DNA MeTase small molecule inhibitor is operablyassociated with the antisense oligonucleotide.

[0013] In a third aspect, the invention provides a method for inhibitingneoplastic cell proliferation in an animal comprising administering toan animal having at least one neoplastic cell present in its body atherapeutically effective amount of an agent of the first aspect of theinvention. In certain preferred embodiments, the agent is an antisenseoligonucleotide which is combined with a pharmaceutically acceptablecarrier and administered for a therapeutically effective period of time.In certain preferred embodiments, the agent is a small moleculeinhibitor which is combined with a pharmaceutically acceptable carrierand administered for a therapeutically effective period of time. Incertain preferred embodiments of the this aspect of the invention, cellproliferation is inhibited in the contacted cell. In preferredembodiments, the cell is a neoplastic cell which may be in an animal,including a human, and which may be in a neoplastic growth. In othercertain embodiments, the agent is a small molecule inhibitor of thefirst aspect of the invention which is combined with a pharmaceuticallyacceptable carrier and administered for a therapeutically effectiveperiod of time. In still yet other preferred embodiments of the thirdaspect of the invention, the method comprises an agent of the firstaspect of the invention which is a combination of one or more antisenseoligonucleotides and/or one or more small molecule inhibitors of thefirst aspect of the invention. In certain preferred embodiments, the DNAMeTase isoform is DNMT-1, DNMT3a or DNMT3b. In other certain preferredembodiments, the DNA MeTase isoform is DNMT3a and/or DNMT3b.

[0014] In a fourth aspect, the invention provides a method foridentifying a specific DNA MeTase isoform that is required for inductionof cell proliferation comprising contacting a cell with an agent of thefirst aspect of the invention. In certain preferred embodiments, theagent is an antisense oligonucleotide that inhibits the expression of aDNA MeTase isoform, wherein the antisense oligonucleotide is specificfor a particular DNA MeTase isoform, and thus inhibition of cellproliferation in the contacted cell identifies the DNA MeTase isoform asa DNA MeTase isoform that is required for induction of cellproliferation. In other certain embodiments, the agent is a smallmolecule inhibitor that inhibits the activity of a DNA MeTase isoform,wherein the small molecule inhibitor is specific for a particular DNAMeTase isoform, and thus inhibition of cell proliferation in thecontacted cell identifies the DNA MeTase isoform as a DNA MeTase isoformthat is required for induction of cell proliferation. In certainpreferred embodiments, the cell is a neoplastic cell, and the inductionof cell proliferation is tumorigenesis. In still yet other preferredembodiments of the fourth aspect of the invention, the method comprisesan agent of the first aspect of the invention which is a combination ofone or more antisense oligonucleotides and/or one or more small moleculeinhibitors of the first aspect of the invention. In certain preferredembodiments, the DNA MeTase isoform is DNMT-1, DNMT3a or DNMT3b. Inother certain preferred embodiments, the DNA MeTase isoform is DNMT3aand/or DNMT3b.

[0015] In a fifth aspect, the invention provides a method foridentifying a DNA MeTase isoform that is involved in induction of celldifferentiation, comprising contacting a cell with an agent thatinhibits the expression of a DNA MeTase isoform, wherein induction ofdifferentiation in the contacted cell identifies the DNA MeTase isoformas a DNA MeTase isoform that is involved in induction of celldifferentiation. In certain preferred embodiments, the agent is anantisense oligonucleotide of the first aspect of the invention. In othercertain preferred embodiments, the agent is a small molecule inhibitorof the first aspect of the invention. In still other certainembodiments, the cell is a neoplastic cell. In still yet other preferredembodiments of the fifth aspect of the invention, the method comprisesan agent of the first aspect of the invention which is a combination ofone or more antisense oligonucleotides and/or one or more small moleculeinhibitors of the first aspect of the invention. In certain preferredembodiments, the DNA MeTase isoform is DNMT-1, DNMT3a or DNMT3b. Inother certain preferred embodiments, the DNA MeTase isoform is DNMT3aand/or DNMT3b. In a sixth aspect, the invention provides a method forinhibiting neoplastic cell growth in an animal comprising administeringto an animal having at least one neoplastic cell present in its body atherapeutically effective amount of an agent of the first aspect of theinvention. In certain embodiments thereof, the agent is an antisenseoligonucleotide, which is combined with a pharmaceutically acceptablecarrier and administered for a therapeutically effective period of time.

[0016] In a seventh aspect, the invention provides a method foridentifying a DNA MeTase isoform that is involved in induction of celldifferentiation, comprising contacting a cell with an antisenseoligonucleotide that inhibits the expression of a DNA MeTase isoform,wherein induction of differentiation in the contacted cell identifiesthe DNA MeTase isoform as a DNA MeTase isoform that is involved ininduction of cell differentiation. Preferably, the cell is a neoplasticcell. In certain preferred embodiments, the DNA MeTase isoform isDNMT-1, DNMT3a or DNMT3b. In other certain preferred embodiments, theDNA MeTase isoform is DNMT3a and/or DNMT3b.

[0017] In an eighth aspect, the invention provides a method forinhibiting cell proliferation in a cell comprising contacting a cellwith at least two agents selected from the group consisting of anantisense oligonucleotide from the first aspect of the invention thatinhibits expression of a specific DNA MeTase isoform, a small moleculeinhibitor from the first aspect of the invention that inhibits aspecific DNA MeTase isoform, an antisense oligonucleotide that inhibitsa DNA methyltransferase, and a small molecule that inhibits a DNAmethyltransferase. In one embodiment, the inhibition of cell growth ofthe contacted cell is greater than the inhibition of cell growth of acell contacted with only one of the agents. In certain embodiments, eachof the agents selected from the group is substantially pure. Inpreferred embodiments, the cell is a neoplastic cell. In yet additionalpreferred embodiments, the agents selected from the group are operablyassociated. In certain preferred embodiments, the DNA MeTase isoform isDNMT-1, DNMT3a or DNMT3b. In other certain preferred embodiments, theDNA MeTase isoform is DNMT3a and/or DNMT3b.

[0018] In a ninth aspect, the invention provides a method for modulatingcell proliferation or differentiation, comprising contacting a cell withan agent of the first aspect of the invention, wherein one or more, butless than all, DNA MeTase isoforms are inhibited, which results in amodulation of proliferation or differentiation. In certain embodiments,the agent is an antisense oligonucleotide of the first aspect of theinvention. In other certain preferred embodiments, the agent is a smallmolecule inhibitor of the first aspect of the invention. In preferredembodiments, the cell proliferation is neoplasia. In still yet otherpreferred embodiments of the this aspect of the invention, the methodcomprises an agent of the first aspect of the invention which is acombination of one or more antisense oligonucleotides and/or one or moresmall molecule inhibitors of the first aspect of the invention. Incertain preferred embodiments, the DNA MeTase isoform is DNMT-1, DNMT3aor DNMT3b. In other certain preferred embodiments, the DNA MeTaseisoform is DNMT3a and/or DNMT3b.

[0019] In an tenth aspect, the invention provides a method forinhibiting cell proliferation in a cell comprising contacting a cellwith at least two agents selected from the group consisting of anantisense oligonucleotide from the first aspect of the invention thatinhibits expression of a specific DNA MeTase isoform, a small moleculeinhibitor that inhibits a specific DNA MeTase isoform, an antisenseoligonucleotide that inhibits a histone deactylase, and a small moleculethat inhibits a histone deactylase. In one embodiment, the inhibition ofcell growth of the contacted cell is greater than the inhibition of cellgrowth of a cell contacted with only one of the agents. In certainembodiments, each of the agents selected from the group is substantiallypure. In preferred embodiments, the cell is a neoplastic cell. In yetadditional preferred embodiments, the agents selected from the group areoperably associated.

[0020] In an eleventh aspect, the invention provides a method forinhibiting cell proliferation in a cell comprising contacting a cellwith at least two agents selected from the group consisting of anantisense oligonucleotide from the first aspect of the invention thatinhibits expression of a specific DNA MeTase isoform, a small moleculeinhibitor that inhibits a specific DNA MeTase isoform, an antisenseoligonucleotide that inhibits a histone deactylase, and a small moleculethat inhibits a histone deactylase. In one embodiment, the inhibition ofcell growth of the contacted cell is greater than the inhibition of cellgrowth of a cell contacted with only one of the agents. In certainembodiments, each of the agents selected from the group is substantiallypure. In preferred embodiments, the cell is a neoplastic cell. In yetadditional preferred embodiments, the agents selected from the group areoperably associated.

BRIEF DESCRIPTION OF THE DRAWINGS

[0021]FIG. 1A is a schematic diagram providing the structures andGenbank accession numbers of the DNA methyltransferase genes, DNMT1,DNMT3a and DNMT3b.

[0022]FIG. 1B is a schematic diagram providing the nucleotide sequenceof DNMT1 cDNA, as provided in GenBank Accession No.(NM_(—)001379).

[0023]FIG. 1C is a schematic diagram providing the nucleotide sequenceof DNMT3a cDNA, as provided in GenBank Accession No.(AF_(—)067972).

[0024]FIG. 1D is a schematic diagram providing the nucleotide sequenceof DNMT3b, as provided in GenBank Accession No. (NM_(—)006892).

[0025]FIG. 1E is a schematic diagram providing the nucleotide sequenceof DNMT3b3, as provided in GenBank Accession No. (AF_(—)156487).

[0026]FIG. 1F is a schematic diagram providing the nucleotide sequenceof DNMT3b4, as provided in GenBank Accession No. (AF_(—)129268).

[0027]FIG. 1G is a schematic diagram providing the nucleotide sequenceof DNMT3b, as provided in GenBank Accession No. (AF_(—)129269).

[0028]FIG. 2 is a schematic diagram providing the structure of theDNMT3a cDNA and the position of antisense oligonucleotides tested ininitial screens. Numbers in parenthesis indicate the starting positionof the antisense oligonucleotides on the DNMT3a sequence. The sequenceand position of the most active antisense inhibitors identified from thescreen is also shown.

[0029]FIG. 3 is a schematic diagram providing the structure of theDNMT3b cDNA and the position of antisense oligonucleotides tested ininitial screens. Numbers in parenthesis indicate the starting positionof the antisense oligonucleotides on the DNMT3b sequence. The sequenceand position of the most active antisense inhibitors identified from thescreen is also shown.

[0030]FIG. 4 is a representation of a Northern blot demonstrating thedose dependent effect of DNMT3a antisense oligonucleotide (SEQ ID NO:33) on the expression of DNMT3a mRNA in A549 human non small cell lungcancer cells. Also demonstrated is the specificity of SEQ ID NO: 33 forDNMT3a as non target mRNAs DNMT1, DNMT3b and Glyceraldehyde 3′-phosphateDehydrogenase are not effected.

[0031]FIG. 5 is a representation of a Northern blot demonstrating thedose dependent effect of DNMT3b antisense oligonucleotide (SEQ ID NO:18) on the expression of DNMT3b mRNA in A549 human non small cell lungcancer cells. Also demonstrated is the specificity of SEQ ID NO: 18 forDNMT3a as non target mRNAs DNMT1, DNMT3a and Glyceraldehyde 3′-phosphateDehydrogenase are not effected.

[0032]FIG. 6 is a representation of a Western blot demonstrating thedose dependent effect of DNMT3b antisense inhibitor SEQ ID NO: 18 on thelevel of DNMT3b protein in T24 human bladder cancer cells and A549 humannon small cell lung cancer cells. Cells were treated for 48 hrs withincreasing doses of SEQ ID NO: 18 after which cells were harvested andDNMT3b levels were determined by Western blot with a DNMT3b specificantibody.

[0033]FIG. 7 is a graphic representation demonstrating the apoptoticeffect of Dnt3a and DNMT3b inhibition on A549 human non small cell lungcancer cells.

[0034]FIG. 8 is a graphic representation demonstrating the Dosedependent apoptotic effect of Dnt3b inhibition on A549 human non smallcell lung cancer cells by three DNMT3b antisense inhibitors.

[0035]FIG. 9 is a graphic representation demonstrating the Dosedependent apoptotic effect of Dnt3b inhibition on T24 human non smallcell lung cancer cells by three DNMT3b antisense inhibitors.

[0036]FIG. 10 is a graphic representation demonstrating the cancerspecific apoptotic effect of DNMT3b inhibition. DNMT3b inhibitor SEQ IDNO: 18 induced apotosis in A549 cells yet similar treatment of the twonormal cell lines HMEC and MRHF produced no apoptosis.

[0037]FIG. 11A is a graphic representation demonstrating the dosedependent effect of Dnmt3b AS1 antisense oligonucleotides on theproliferation of human A549 cancer cells.

[0038]FIG. 11B is a graphic representation demonstrating the cancerspecificity of antiproliferative effect of Dnmt3a and Dnmt3b inhibition.Inhibition of Dnmt3a or Dnmt3b produces antiproliferative effects ofcancer cells but not affect the proliferation of the human normal skinfibroblast cell line MRHF.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0039] The invention provides methods and agents for inhibiting specificDNA MeTase isoforms by inhibiting expression at the nucleic acid levelor protein activity at the enzymatic level. The invention allows theidentification of and specific inhibition of specific DNA MeTaseisoforms involved in tumorigenesis and thus provides a treatment forcancer. The invention further allows identification of and specificinhibition of specific DNA MeTase isoforms involved in cellproliferation and/or differentiation and thus provides a treatment forcell proliferative and/or differentiation disorders.

[0040] The patent and scientific literature referred to hereinestablishes knowledge that is available to those with skill in the art.The issued patents, applications, and references, including GenBankdatabase sequences, that are cited herein are hereby incorporated byreference to the same extent as if each was specifically andindividually indicated to be incorporated by reference.

[0041] In a first aspect, the invention provides agents that inhibit oneor more DNA MeTase isoforms, but less than all specific DNA MeTaseisoforms. As used herein interchangeably, the terms “DNA MeTase”,“DNMT”, “DNA MeTase isoform”, “DNMT isoform” and similar terms areintended to refer to any one of a family of enzymes that add a methylgroups to the C5 position of cytosine in DNA. Preferred DNA MeTaseisoforms include maintenance and de novo methyltransferases. SpecificDNA MeTases include without limitation, DNMT-1, DNMT3a, and DNMT3b. Byway of non-limiting example, useful agents that inhibit one or more DNAMeTase isoforms, but less than all specific DNA MeTase isoforms, includeantisense oligonucleotides and small molecule inhibitors.

[0042] The present inventors have surprisingly discovered that specificinhibition of DNMT-1 reverses the tumorigenic state of a transformedcell. The inventors have also surprisingly discovered that theinhibition of the DNMT3b and/or DNMT3b isoform dramatically inducesgrowth arrest and apoptosis in cancerous cells. Thus, in certainembodiments of this aspect of the invention, the DNA MeTase isoform thatis inhibited is DNMT3a and/or DNMT3b.

[0043] Preferred agents that inhibit DNMT3a and/or DNMT3b dramaticallyinhibit growth of human cancer cells, independent of p53 status. Theseagents significantly induce apoptosis in the cancer cells and causedramatic growth arrest. Inhibitory agents that achieve one or more ofthese results are considered within the scope of this aspect of theinvention. By way of non-limiting example, antisense oligonucleotidesand/or small molecule inhibitors of DNMT3a and/or DNMT3b are useful forthe invention.

[0044] In certain preferred embodiments, the agent that inhibits thespecific DNMT isoform is an oligonucleotide that inhibits expression ofa nucleic acid molecule encoding a specific DNA MeTase isoform. Thenucleic acid molecule may be genomic DNA (e.g., a gene), cDNA, or RNA.In other embodiments, the oligonucleotide ultimately inhibitstranslation of the DNA MeTase. In certain embodiments theoligonucleotide causes the degradation of the nucleic acid molecule.Preferred antisense oligonucleotides have potent and specific antisenseactivity at nanomolar concentrations.

[0045] The antisense oligonucleotides according to the invention arecomplementary to a region of RNA or double-stranded DNA that encodes aportion of one or more DNA MeTase isoforms (taking into account thathomology between different isoforms may allow a single antisenseoligonucleotide to be complementary to a portion of more than oneisoform).

[0046] For purposes of the invention, the term “complementary” meanshaving the ability to hybridize to a genomic region, a gene, or an RNAtranscript thereof under physiological conditions. Such hybridization isordinarily the result of base-specific hydrogen bonding betweencomplementary strands, preferably to form Watson-Crick or Hoogsteen basepairs, although other modes of hydrogen bonding, as well as basestacking can lead to hybridization. As a practical matter, suchhybridization can be inferred from the observation of specific geneexpression inhibition, which may be at the level of transcription ortranslation (or both).

[0047] For purposes of the invention, the term “oligonucleotide”includes polymers of two or more deoxyribonucleosides, ribonucleosides,or 2′-O-substituted ribonucleoside residues, or any combination thereof.Preferably, such oligonucleotides have from about 8 to about 50nucleoside residues, and most preferably from about 12 to about 30nucleoside residues. The nucleoside residues may be coupled to eachother by any of the numerous known internucleoside linkages. Suchinternucleoside linkages include without limitation phosphorothioate,phosphorodithioate, alkylphosphonate, alkylphosphonothioate,phosphotriester, phosphoramidate, siloxane, carbonate,carboxymethylester, acetamidate, carbamate, thioether, bridgedphosphoramidate, bridged methylene phosphonate, bridgedphosphorothioate, and sulfone internucleotide linkages. In certainpreferred embodiments, these internucleoside linkages may bephosphodiester, phosphotriester, phosphorothioate, or phosphoramidatelinkages, or combinations thereof. The term oligonucleotide alsoencompasses such polymers having chemically modified bases or sugarsand/or having additional substituents, including without limitationlipophilic groups, intercalating agents, diamines, and adamantane. Theterm oligonucleotide also encompasses such polymers as PNA and LNA. Forpurposes of the invention the term “2′-O-substituted” means substitutionof the 2′ position of the pentose moiety with an -O-lower alkyl groupcontaining 1-6 saturated or unsaturated carbon atoms, or with an -O-arylor allyl group having 2-6 carbon atoms, wherein such alkyl, aryl, orallyl group may be unsubstituted or may be substituted, e.g., with halo,hydroxy, trifluoromethyl, cyano, nitro, acyl, acyloxy, alkoxy, carboxyl,carbalkoxyl, or amino groups; or such 2′ substitution may be with ahydroxy group (to produce a ribonucleoside), an amino or a halo group,but not with a 2′-H group.

[0048] Particularly preferred antisense oligonucleotides utilized inthis aspect of the invention include chimeric oligonucleotides andhybrid oligonucleotides.

[0049] For purposes of the invention, a “chimeric oligonucleotide”refers to an oligonucleotide having more than one type ofinternucleoside linkage. One preferred embodiment of such a chimericoligonucleotide is a chimeric oligonucleotide comprising aphosphorothioate, phosphodiester or phosphorodithioate region,preferably comprising from about 2 to about 12 nucleotides, and analkylphosphonate or alkylphosphonothioate region (see e.g., Pederson etal. U.S. Pat. Nos. 5,635,377 and 5,366,878). Preferably, such chimericoligonucleotides contain at least three consecutive internucleosidelinkages selected from phosphodiester and phosphorothioate linkages, orcombinations thereof.

[0050] For purposes of the invention, a “hybrid oligonucleotide” refersto an oligonucleotide having more than one type of nucleoside. Onepreferred embodiment of such a hybrid oligonucleotide comprises aribonucleotide or 2′-O-substituted ribonucleotide region, preferablycomprising from about 2 to about 12 2′-O-substituted nucleotides, and adeoxyribonucleotide region. Preferably, such a hybrid oligonucleotidewill contain at least three consecutive deoxyribonucleosides and willalso contain ribonucleosides, 2′-O-substituted ribonucleosides, orcombinations thereof (see e.g., Metelev and Agrawal, U.S. Pat. Nos.5,652,355 and 5,652,356).

[0051] The exact nucleotide sequence and chemical structure of anantisense oligonucleotide utilized in the invention can be varied, solong as the oligonucleotide retains its ability to inhibit expression ofa specific DNA MeTase isoform or inhibit one or more DNA MeTaseisoforms, but less than all specific DNA MeTase isoforms. This isreadily determined by testing whether the particular antisenseoligonucleotide is active by quantitating the amount of mRNA encoding aspecific DNA MeTase isoform, quantitating the amount of DNA MeTaseisoform protein, quantitating the DNA MeTase isoform enzymatic activity,or quantitating the ability of the DNA MeTase isoform to inhibit cellgrowth in a an in vitro or in vivo cell growth assay, all of which aredescribed in detail in this specification. The term “inhibit expression”and similar terms used herein are intended to encompass any one or moreof these parameters.

[0052] Antisense oligonucleotides utilized in the invention mayconveniently be synthesized on a suitable solid support using well-knownchemical approaches, including H-phosphonate chemistry, phosphoramiditechemistry, or a combination of H-phosphonate chemistry andphosphoramidite chemistry (i.e., H-phosphonate chemistry for some cyclesand phosphoramidite chemistry for other cycles). Suitable solid supportsinclude any of the standard solid supports used for solid phaseoligonucleotide synthesis, such as controlled-pore glass (CPG) (see,e.g., Pon, R. T., Methods in Molec. Biol. 20: 465-496, 1993).

[0053] Antisense oligonucleotides according to the invention are usefulfor a variety of purposes. For example, they can be used as “probes” ofthe physiological function of specific DNA MeTase isoforms by being usedto inhibit the activity of specific DNA MeTase isoforms in anexperimental cell culture or animal system and to evaluate the effect ofinhibiting such specific DNA MeTase isoform activity. This isaccomplished by administering to a cell or an animal an antisenseoligonucleotide that inhibits the expression of one or more DNA MeTaseisoforms according to the invention and observing any phenotypiceffects. In this use, the antisense oligonucleotides according to theinvention is preferable to traditional “gene knockout” approachesbecause it is easier to use, and can be used to inhibit specific DNAMeTase isoform activity at selected stages of development ordifferentiation.

[0054] Preferred antisense oligonucleotides of the invention inhibiteither the transcription of a nucleic acid molecule encoding the DNAMeTase isoform, and/or the translation of a nucleic acid moleculeencoding the DNA MeTase isoform, and/or lead to the degradation of suchnucleic acid. DNA MeTase-encoding nucleic acids may be RNA or doublestranded DNA regions and include, without limitation, intronicsequences, untranslated 5′ and 3′ regions, intron-exon boundaries aswell as coding sequences from a DNA MeTase family member gene. (See,e.g., Yoder, J. A., et al. (1996) J. Biol. Chem. 271:31092-31097; Xie,S., et al. (1999) Gene 236:87-95; and Robertson, K. D., et al. (1999)Nucleic Acids Research 27:2291-2298).

[0055] Particularly preferred non-limiting examples of antisenseoligonucleotides of the invention are complementary to regions of RNA ordouble-stranded DNA encoding a DNA MeTase isoform (e.g., DNMT-1, DNMT3a,DNMT3b (also known as DNMT3b1), DNMT3b2, DNMT3b3, DNMT3b3, DNMT3b4,DNMT3b5). (see e.g., GenBank Accession No. NM_(—)001379 for human DNMT-1(FIG. 1B); GenBank Accession No. AF_(—)067972 for human DNMT3a, (FIG.1C); GenBank Accession Nos. NM_(—)006892, AF_(—)156488, AF_(—)176228,and XM_(—)009449 for human DNMT3b (FIG. 1D); nucleotide positions115-1181 and 1240-2676 of GenBank No. NM_(—)006892 for human DNMT3b2,GenBank Accession No. AF_(—)156487 for human DNMT3b3 (FIG. 1E), GenBankAccession No. AF_(—)129268 for human DNMT3b4 (FIG. 1F), and GenBankAccession No. AF_(—)129269 for human DNMT3b5 (FIG. 1G).

[0056] As used herein, a reference to any one of the specific DNAMeTases isoforms includes reference to all RNA splice variants of thatparticular isoform. By way of non-limiting example, reference to DNMT3bis meant to include the splice variants DNMTb2, DNMTb3, DNMTb4, andDNMTb5.

[0057] The sequences encoding DNA MeTases from non-human animal speciesare also known (see, for example, GenBank Accession Numbers AF_(—)175432(murine DNMT-1); NM_(—)010068 (murine DNMT3a); and NM_(—)007872 (murineDNMT3b). Accordingly, the antisense oligonucleotides of the inventionmay also be complementary to regions of RNA or double-stranded DNA thatencode DNA MeTases from non-human animals. Antisense oligonucleotidesaccording to these embodiments are useful as tools in animal models forstudying the role of specific DNA MeTase isoforms.

[0058] Particularly, preferred oligonucleotides have nucleotidesequences of from about 13 to about 35 nucleotides which include fromabout 13 to all of a nucleotide sequence shown in Table 1 and Table 2.Yet additional particularly preferred oligonucleotides have nucleotidesequences of from about 15 to about 26 nucleotides. Most preferably, theoligonucleotides shown below have phosphorothioate backbones, are 20-26nucleotides in length, and are modified such that the terminal fournucleotides at the 5′ end of the oligonucleotide and the terminal fournucleotides at the 3′ end of the oligonucleotide each have 2′-O-methylgroups attached to their sugar residues.

[0059] Antisense oligonucleotides used in the present study are shown inTable 1 and Table 2. TABLE 1 Sequences of Human DNA MeTase DNMT1Antisense (AS) Oligonucleotides and Their Mismatch (MM) Oligonucleotides(SEQ (SEQ ID IC₅₀ ID IC₅₀ Sequence NO) (nM)¹ NO) (nM)²5′CAGGTAGCCCTCCTCGGAT 03′ [4] 90 [11] 70 5′AAGCATGAGCACCGTTCTCC 3′ [5]66 [12] 43 5′TTCATGTCAGCCAAGGCCAC 3′ [6] 67 [13] 605′CGAACCTCACACAACAGCTT 3′ [7] 96 [14] 75 5′GATAAGCGAACCTCACACAA 3′ [8]90 [15] 81 5′CCAAGGCCACAAACACCATG 3′ [9] 66 [16] 605′CATCTGCCATTCCCACTCTA 3′ [10]³ 133 [17] 114 Scrambled sequence -- >>250-- >>250

[0060] TABLE 2 Sequences of Human DNA MeTase DNMT3a and DNMT3b Antisense(AS) Oligonucleotides and Their Mismatch (MM) OligonucleotidesNucleotide Target Accession Number Position Chemistry Sequence DNMT3B ASNM_006892 3′UTR (3993) PTI 5′cgtcgtggctccagttacaa3′ (SEQ ID NO:18)DNMT3B MM NM_006892 PTI 5′cctcgtcggtcgacttagaa3′ (SEQ ID NO:19) DNMT3BAS NM_006892 3′UTR (3993) PTI-Ome 5′cgucgtggctccagttacaa3′ (SEQ IDNO:20) DNMT3B MM NM_006892 PTI-Ome 5′ccucgtcggtcgacttagaa3′ (SEQ IDNO:21) DNMT3B AS NM_006892 3′UTR (3023) PTI 5′agagctgtcggcactgtggt3′(SEQ ID NO:22) DNMT3B AS NM_006892 3′UTR (3023) PTI-Ome5′agagctgtcggcactguggu3′ (SEQ ID NO:23) DNMT3B MM NM_006892 PTI-Ome5′acaggtgtggccagtgucgu3′ (SEQ ID NO:24) DNMT3B AS NM_008892 3′UTR (3997)PTI 5′tgttacgtcgtggctccagt3′ (SEQ ID NO:25) DNMT3B AS NM_006892 3′UTR(3997) PTI-Ome 5′uguuacgtcgtggctccagu3′ (SEQ ID NO:26) DNMT3B MMNM_006892 PTI-Ome 5′ucuuaggtcctgcctgcacu3′ (SEQ ID NO:27) DNMT3A ASAF067972.1 3′UTR (3258) PTI 5′tgatgtccaaccctttucgc3′ (SEQ ID NO:28)DNMT3A AS AF067972.1 3′UTR (3258) PTI-Ome 5′ugaugtccaaccctttucgc3′ (SEQID NO:29) DNMT3A AS AP067972.1 3′UTR (3434) PTI 5′caggagatgatgtccaaccc3′(SEQ ID NO:30) DNMT3A AS AF067972.1 3′UTR (3434) PTI-Ome5′caggagatgatgtccaaccc3′ (SEQ ID NO:31) DNMT3A MM AF067972.1 PTI-Ome5′cacgacatcatctcgaacgc3′ (SEQ ID NO:32) DNMT3A AS AF067972.1 3′UTR(4045) PTI 5′cgtgagaacgcgccatctgc3′ (SEQ ID NO:33) DNMT3A AS AF067972.13′UTR (4045) PTI-Ome 5′cgugagaacgcgccatcugc3′ (SEQ ID NO:34) DNMT3A MMAF067972.1 PTI-Ome 5′ccugacaaggcccgatgugc3′ (SEQ ID NO:35) DNMT3A ASAF067972.1 3′UTR (4302) PTI 5′gttctgatcccaccacaagg3′ (SEQ ID NO:36)DNMT3A AS AF067972.1 3′UTR (4302) PTI-Ome 5′guuctgatcccaccacaagg3′ (SEQID NO:37)

[0061] The antisense oligonucleotides according to the invention mayoptionally be formulated with any of the well known pharmaceuticallyacceptable carriers or diluents (see preparation of pharmaceuticallyacceptable formulations in, e.g., Remington's Pharmaceutical Sciences,18th Edition, ed. A. Gennaro, Mack Publishing Co., Easton, Pa., 1990),with the proviso that such carriers or diluents not affect their abilityto modulate DNA MeTase activity.

[0062] By way of non-limiting example, the agent of the first aspect ofthe invention may also be a small molecule inhibitor. The term “smallmolecule” as used in reference to the inhibition of DNA MeTase is usedto identify a compound having a molecular weight preferably less than1000 Da, more preferably less than 800 Da, and most preferably less than600 Da, which is capable of interacting with a DNA MeTase and inhibitingthe expression of a nucleic acid molecule encoding an DNMT isoform oractivity of an DNMT protein. Inhibiting DNA MeTase enzymatic activitymeans reducing the ability of a DNA MeTase to add a methyl group to theC5 position of cytosine. In some preferred embodiments, such reductionof DNA MeTase activity is at least about 50%, more preferably at leastabout 75%, and still more preferably at least about 90%. In otherpreferred embodiments, DNA MeTase activity is reduced by at least 95%and more preferably by at least 99%. In one certain embodiment, thesmall molecule inhibitor is an inhibitor of one or more but less thanall DNMT isoforms. By “all DNMT isoforms” is meant all proteins thatspecifically add a methyl group to the C5 position of cytosine, andincludes, without limitation, DNMT-1, DNMT3a, or DNMT3b, all of whichare considered “related proteins,” as used herein.

[0063] Most preferably, a DNA MeTase small molecule inhibitor interactswith and reduces the activity of one or more DNA MeTase isoforms (e.g.,DNMT3a and/or DNMT3b), but does not interact with or reduce theactivities of all of the other DNA MeTase isoforms (e.g., DNMT-1, DNMT3aand DNMT3b). As discussed below, a preferred DNA MeTase small moleculeinhibitor is one that interacts with and reduces the enzymatic activityof a DNA MeTase isoform that is involved in tumorigenesis.

[0064] The invention disclosed herein encompasses the use of differentlibraries for the identification of small molecule inhibitors of one ormore, but not all, MeTases. Libraries useful for the purposes of theinvention include, but are not limited to, (1) chemical libraries, (2)natural product libraries, and (3) combinatorial libraries comprised ofrandom peptides, oligonucleotides and/or organic molecules.

[0065] Chemical libraries consist of structural analogs of knowncompounds or compounds that are identified as “hits” or “leads” vianatural product screening. Natural product libraries are derived fromcollections of microorganisms, animals, plants, or marine organismswhich are used to create mixtures for screening by: (1) fermentation andextraction of broths from soil, plant or marine microorganisms or (2)extraction of plants or marine organisms. Natural product librariesinclude polyketides, non-ribosomal peptides, and variants (non-naturallyoccurring) thereof. For a review, see , Cane, D. E., et al., (1998)Science 282:63-68. Combinatorial libraries are composed of large numbersof peptides, oligonucleotides or organic compounds as a mixture. Theyare relatively easy to prepare by traditional automated synthesismethods, PCR, cloning or proprietary synthetic methods. Of particularinterest are peptide and oligonucleotide combinatorial libraries.

[0066] More specifically, a combinatorial chemical library is acollection of diverse chemical compounds generated by either chemicalsynthesis or biological synthesis, by combining a number of chemical“building blocks” such as reagents. For example, a linear combinatorialchemical library such as a polypeptide library is formed by combining aset of chemical building blocks (amino acids) in every possible way fora given compound length (i.e., the number of amino acids in apolypeptide compound). Millions of chemical compounds can be synthesizedthrough such combinatorial mixing of chemical building blocks.

[0067] For a review of combinatorial chemistry and libraries createdtherefrom, see Huc, I. and Nguyen, R. (2001) Comb. Chem. High ThroughputScreen 4:53-74; Lepre, C. A. (2001) Drug Discov. Today 6:133-140; Peng,S. X. (2000) Biomed. Chromatogr. 14:430-441; Bohm, H. J. and Stahl, M.(2000) Curr. Opin. Chem. Biol. 4:283-286; Barnes, C. andBalasubramanian, S. (2000) Curr. Opin. Chem. Biol. 4:346-350; Lepre,Enjalbal, C., et al., (2000) Mass Septrom Rev. 19:139-161; Hall, D. G.,(2000) Nat. Biotechnol. 18:262-262; Lazo, J. S., and Wipf, P. (2000) J.Pharmacol. Exp. Ther. 293:705-709; Houghten, R. A., (2000) Ann. Rev.Pharmacol. Toxicol. 40:273-282; Kobayashi, S. (2000) Curr. Opin. Chem.Biol. (2000) 4:338-345; Kopylov, A. M. and Spiridonova, V. A. (2000)Mol. Biol. (Mosk) 34:1097-1113; Weber, L. (2000) Curr. Opin. Chem. Biol.4:295-302; Dolle, R. E. (2000) J. Comb. Chem. 2:383-433; Floyd, C. D.,et al., (1999) Prog. Med. Chem. 36:91-168; Kundu, B., et al., (1999)Prog. Drug Res. 53:89-156; Cabilly, S. (1999) Mol. Biotechnol.12:143-148; Lowe, G. (1999) Nat. Prod. Rep. 16:641-651; Dolle, R. E. andNelson, K. H. (1999) J. Comb. Chem. 1:235-282; Czarnick, A. W. andKeene, J. D. (1998) Curr. Biol. 8:R705-R707; Dolle, R. E. (1998) Mol.Divers. 4:233-256; Myers, P. L., (1997) Curr. Opin. Biotechnol.8:701-707; and Pluckthun, A. and Cortese, R. (1997) Biol. Chem. 378:443.

[0068] Devices for the preparation of combinatorial libraries arecommercially available (see, e.g., 357 MPS, 390 MPS, Advanced Chem Tech,Louisville Ky., Symphony, Rainin, Woburn, Mass., 433A AppliedBiosystems, Foster City, Calif., 9050 Plus, Millipore, Bedford, Mass.).In addition, numerous combinatorial libraries are themselvescommercially available (see, e.g., ComGenex, Princeton, N.J., Asinex,Moscow, Ru, Tripos, Inc., St. Louis, Mo., ChemStar, Ltd., Moscow, RU, 3DPharmaceuticals, Exton, Pa., Martek Biosciences, Columbia, Md., etc.).

[0069] Still other libraries of interest include peptide, protein,peptidomimetic, multiparallel synthetic collection, recombinatorial, andpolypeptide libraries.

[0070] Small molecule inhibitors of one or more, but not all, MeTasesare identified and isolated from the libraries described herein by anymethod known in the art. Such screening methods include, but are notlimited to, functional screening and affinity binding methodologies. Inaddition, the screening methods utilized for the identification of smallmolecule inhibitors of one or more, but not all, MeTases include highthroughput assays. By way of non-limiting example, Meldal, M. disclosesthe use of combinatorial solid-phase assays for enzyme activity andinhibition experiments (Meldal, M. (1998) Methods Mol. Biol. 87:51-57),and Dolle, R. E. describes generally the use of combinatorial librariesfor the discovery of inhibitors of enzymes (Dolle, R. E. (1997) Mol.Divers. 2:223-236).

[0071] By way of non-limiting example, Example 5 below provides a smallmolecule inhibitor screen encompassed by the invention.

[0072] The agents according to the invention are useful as analyticaltools and as therapeutic tools, including as gene therapy tools. Theinvention also provides methods and compositions which may bemanipulated and fine-tuned to fit the condition(s) to be treated whileproducing fewer side effects.

[0073] In a second aspect, the invention provides a method forinhibiting one or more, but less than all, DNA MeTase isoforms in a cellcomprising contacting the cell with an agent of the first aspect of theinvention. By way of non-limiting example, the agent may be an antisenseoligonucleotide or a small molecule inhibitor that inhibits theexpression of one or more, but less than all, specific DNA MeTaseisoforms in the cell.

[0074] In certain embodiments, the invention provides a methodcomprising contacting a cell with an antisense oligonucleotide thatinhibits one or more but less than all DNA MeTase isoforms in the cell.Preferably, cell proliferation is inhibited in the contacted cell. Thus,the antisense oligonucleotides according to the invention are useful intherapeutic approaches to human diseases, including benign and malignantneoplasms, by inhibiting cell proliferation in cells contacted with theantisense oligonucleotides. The phrase “inhibiting cell proliferation”is used to denote an ability of a DNA MeTase antisense oligonucleotideor a small molecule DNA MeTase inhibitor (or combination thereof) toretard the growth of cells contacted with the oligonucleotide or smallmolecule inhibitor, as compared to cells not contacted. Such anassessment of cell proliferation can be made by counting contacted andnon-contacted cells using a Coulter Cell Counter (Coulter, Miami, Fla.)or a hemacytometer. Where the cells are in a solid growth (e.g., a solidtumor or organ), such an assessment of cell proliferation can be made bymeasuring the growth with calipers, and comparing the size of the growthof contacted cells with non-contacted cells. Preferably, the termincludes a retardation of cell proliferation that is at least 50%greater than non-contacted cells. More preferably, the term includes aretardation of cell proliferation that is 100% of non-contacted cells(i.e., the contacted cells do not increase in number or size). Mostpreferably, the term includes a reduction in the number or size ofcontacted cells, as compared to non-contacted cells. Thus, a DNA MeTaseantisense oligonucleotide or a DNA MeTase small molecule inhibitor thatinhibits cell proliferation in a contacted cell may induce the contactedcell to undergo growth retardation, to undergo growth arrest, to undergoprogrammed cell death (i.e., to apoptose), or to undergo necrotic celldeath.

[0075] Conversely, the phrase “inducing cell proliferation” and similarterms are used to denote the requirement of the presence or enzymaticactivity of a specific DNA MeTase isoform for cell proliferation in anormal (i.e., non-neoplastic) cell. Hence, over-expression of a specificDNA MeTase isoform that induces cell proliferation may or may not leadto increased cell proliferation; however, inhibition of a specific DNAMeTase isoform that induces cell proliferation will lead to inhibitionof cell proliferation.

[0076] The cell proliferation inhibiting ability of the antisenseoligonucleotides according to the invention allows the synchronizationof a population of a-synchronously growing cells. For example, theantisense oligonucleotides of the invention may be used to arrest apopulation of non-neoplastic cells grown in vitro in the G1 or G2 phaseof the cell cycle. Such synchronization allows, for example, theidentification of gene and/or gene products expressed during the G1 orG2 phase of the cell cycle. Such a synchronization of cultured cells mayalso be useful for testing the efficacy of a new transfection protocol,where transfection efficiency varies and is dependent upon theparticular cell cycle phase of the cell to be transfected. Use of theantisense oligonucleotides of the invention allows the synchronizationof a population of cells, thereby aiding detection of enhancedtransfection efficiency.

[0077] The anti-neoplastic utility of the antisense oligonucleotidesaccording to the invention is described in detail elsewhere in thisspecification.

[0078] In yet other preferred embodiments, the cell contacted with a DNAMeTase antisense oligonucleotide is also contacted with a DNA MeTasesmall molecule inhibitor.

[0079] In a few preferred embodiments, the DNA MeTase small moleculeinhibitor is operably associated with the antisense oligonucleotide. Asmentioned above, the antisense oligonucleotides according to theinvention may optionally be formulated with well known pharmaceuticallyacceptable carriers or diluents. This formulation may further containone or more one or more additional DNA MeTase antisenseoligonucleotide(s), and/or one or more DNA MeTase small moleculeinhibitor(s), or it may contain any other pharmacologically activeagent.

[0080] In a particularly preferred embodiment of the invention, theantisense oligonucleotide is in operable association with a DNA MeTasesmall molecule inhibitor. The term “operable association” includes anyassociation between the antisense oligonucleotide and the DNA MeTasesmall molecule inhibitor which allows an antisense oligonucleotide toinhibit the expression of one or more specific DNA MeTaseisoform-encoding nucleic acids and allows the DNA MeTase small moleculeinhibitor to inhibit specific DNA MeTase isoform enzymatic activity. Oneor more antisense oligonucleotides of the invention may be operablyassociated with one or more DNA MeTase small molecule inhibitors. Insome preferred embodiments, an antisense oligonucleotide of theinvention that targets one particular DNA MeTase isoform (e.g., DNMT-1,DNMT3a, or DNMT3b) is operably associated with a DNA MeTase smallmolecule inhibitor which targets the same DNA MeTase isoform. Apreferred operable association is hydrolyzable. Preferably, thehydrolyzable association is a covalent linkage between the antisenseoligonucleotide and the DNA MeTase small molecule inhibitor. Preferably,such covalent linkage is hydrolyzable by esterases and/or amidases.Examples of such hydrolyzable associations are well known in the art.Phosphate esters are particularly preferred.

[0081] In certain preferred embodiments, the covalent linkage may bedirectly between the antisense oligonucleotide and the DNA MeTase smallmolecule inhibitor so as to integrate the DNA MeTase small moleculeinhibitor into the backbone. Alternatively, the covalent linkage may bethrough an extended structure and may be formed by covalently linkingthe antisense oligonucleotide to the DNA MeTase small molecule inhibitorthrough coupling of both the antisense oligonucleotide and the DNAMeTase small molecule inhibitor to a carrier molecule such as acarbohydrate, a peptide or a lipid or a glycolipid. Other preferredoperable associations include lipophilic association, such as formationof a liposome containing an antisense oligonucleotide and the DNA MeTasesmall molecule inhibitor covalently linked to a lipophilic molecule andthus associated with the liposome. Such lipophilic molecules includewithout limitation phosphotidylcholine, cholesterol,phosphatidylethanolamine, and synthetic neoglycolipids, such assyalyllacNAc-HDPE. In certain preferred embodiments, the operableassociation may not be a physical association, but simply a simultaneousexistence in the body, for example, when the antisense oligonucleotideis associated with one liposome and the small molecule inhibitor isassociated with another liposome.

[0082] In a third aspect, the invention provides a method for inhibitingneoplastic cell proliferation in an animal comprising administering toan animal having at least one neoplastic cell present in its body atherapeutically effective amount of an agent of the first aspect of theinvention. In one certain embodiment, the agent is an antisenseoligonucleotide of the first aspect of the invention, and the methodfurther comprises a pharmaceutically acceptable carrier. The antisenseoligonucleotide and the pharmaceutically acceptable carrier areadministered for a therapeutically effective period of time. Preferably,the animal is a mammal, particularly a domesticated mammal. Mostpreferably, the animal is a human.

[0083] The term “neoplastic cell” is used to denote a cell that showsaberrant cell growth. Preferably, the aberrant cell growth of aneoplastic cell is increased cell growth. A neoplastic cell may be ahyperplastic cell, a cell that shows a lack of contact inhibition ofgrowth in vitro, a benign tumor cell that is incapable of metastasis invivo, or a cancer cell that is capable of metastases in vivo and thatmay recur after attempted removal. The term “tumorigenesis” is used todenote the induction of cell proliferation that leads to the developmentof a neoplastic growth.

[0084] The terms “therapeutically effective amount” and “therapeuticallyeffective period of time” are used to denote known treatments at dosagesand for periods of time effective to reduce neoplastic cell growth.Preferably, such administration should be parenteral, oral, sublingual,transdermal, topical, intranasal, or intrarectal. When administeredsystemically, the therapeutic composition is preferably administered ata sufficient dosage to attain a blood level of antisense oligonucleotidefrom about 0.1 μM to about 10 μM. For localized administration, muchlower concentrations than this may be effective, and much higherconcentrations may be tolerated. One of skill in the art will appreciatethat such therapeutic effect resulting in a lower effectiveconcentration of the DNA MeTase inhibitor may vary considerablydepending on the tissue, organ, or the particular animal or patient tobe treated according to the invention.

[0085] In a preferred embodiment, the therapeutic composition of theinvention is administered systemically at a sufficient dosage to attaina blood level of antisense oligonucleotide from about 0.01 μM to about20 μM. In a particularly preferred embodiment, the therapeuticcomposition is administered at a sufficient dosage to attain a bloodlevel of antisense oligonucleotide from about 0.05 μM to about 15 μM. Ina more preferred embodiment, the blood level of antisenseoligonucleotide is from about 0.1 μM to about 10 μM.

[0086] For localized administration, much lower concentrations than thismay be therapeutically effective. Preferably, a total dosage ofantisense oligonucleotide will range from about 0.1 mg to about 200 mgoligonucleotide per kg body weight per day. In a more preferredembodiment, a total dosage of antisense oligonucleotide will range fromabout 1 mg to about 20 mg oligonucleotide per kg body weight per day. Ina most preferred embodiment, a total dosage of antisense oligonucleotidewill range from about 1 mg to about 10 mg oligonucleotide per kg bodyweight per day. In a particularly preferred embodiment, thetherapeutically effective amount of a DNA MeTase antisenseoligonucleotide is about 5 mg oligonucleotide per kg body weight perday.

[0087] In certain preferred embodiments of the third aspect of theinvention, the method further comprises administering to the animal atherapeutically effective amount of a DNA MeTase small moleculeinhibitor with a pharmaceutically acceptable carrier for atherapeutically effective period of time. In some preferred embodiments,the DNA MeTase small molecule inhibitor is operably associated with theantisense oligonucleotide, as described supra.

[0088] The DNA MeTase small molecule inhibitor-containing therapeuticcomposition of the invention is administered systemically at asufficient dosage to attain a blood level DNA MeTase small moleculeinhibitor from about 0.01 μM to about 10 μM. In a particularly preferredembodiment, the therapeutic composition is administered at a sufficientdosage to attain a blood level of DNA MeTase small molecule inhibitorfrom about 0.05 μM to about 10 μM. In a more preferred embodiment, theblood level of DNA MeTase small molecule inhibitor is from about 0.1 μMto about 5 μM. For localized administration, much lower concentrationsthan this may be effective. Preferably, a total dosage of DNA MeTasesmall molecule inhibitor will range from about 0.01 mg to about 100 mgprotein effector per kg body weight per day. In a more preferredembodiment, a total dosage of DNA MeTase small molecule inhibitor willrange from about 0.1 mg to about 50 mg protein effector per kg bodyweight per day. In a most preferred embodiment, a total dosage of DNAMeTase small molecule inhibitor will range from about 0.1 mg to about 10mg protein effector per kg body weight per day. In a particularlypreferred embodiment, the therapeutically effective synergistic amountof DNA MeTase small molecule inhibitor (when administered with anantisense oligonucleotide) is about 5 mg per kg body weight per day.

[0089] Certain preferred embodiments of this aspect of the inventionresult in an improved inhibitory effect, thereby reducing thetherapeutically effective concentrations of either or both of thenucleic acid level inhibitor (i.e., antisense oligonucleotide) and theprotein level inhibitor (i.e., DNA MeTase small molecule inhibitor)required to obtain a given inhibitory effect as compared to thosenecessary when either is used individually.

[0090] Furthermore, one of skill will appreciate that thetherapeutically effective synergistic amount of either the antisenseoligonucleotide or the DNA MeTase inhibitor may be lowered or increasedby fine tuning and altering the amount of the other component. Theinvention therefore provides a method to tailor theadministration/treatment to the particular exigencies specific to agiven animal species or particular patient. Therapeutically effectiveranges may be easily determined for example empirically by starting atrelatively low amounts and by step-wise increments with concurrentevaluation of inhibition.

[0091] In a fourth aspect, the invention provides a method foridentifying a specific DNA MeTase isoform that is required for inductionof cell proliferation comprising contacting a growing cell with an agentof the first aspect of the invention. In certain preferred embodiments,the agent is an antisense oligonucleotide that inhibits the expressionof a DNA MeTase isoform, wherein the antisense oligonucleotide isspecific for a particular DNMT isoform, and thus inhibition of cellproliferation in the contacted cell identifies the DNA MeTase isoform asa DNA MeTase isoform that is required for induction of cellproliferation. In other certain embodiments, the agent is a smallmolecule inhibitor that inhibits the activity of a DNA MeTase isoform,wherein the small molecule inhibitor is specific for a particular DNMTisoform, and thus inhibition of cell proliferation in the contacted cellidentifies the DNA MeTase isoform as a DNA MeTase isoform that isrequired for induction of cell proliferation. In certain preferredembodiments, the cell is a neoplastic cell, and the induction of cellproliferation is tumorigenesis. In still yet other preferred embodimentsof the fourth aspect of the invention, the method comprises an agent ofthe first aspect of the invention which is a combination of one or moreantisense oligonucleotides and/or one or more small molecule inhibitorsof the first aspect of the invention. In certain preferred embodiments,the DNA MeTase isoform is DNMT-1, DNMT3a, or DNMT3b. In other certainpreferred embodiments, the DNA MeTase isoform is DNMT3a and/or DNMT3b.

[0092] In a fifth aspect, the invention provides a method foridentifying a DNA MeTase isoform that is involved in induction of celldifferentiation comprising contacting a cell with an agent that inhibitsthe expression of a DNA MeTase isoform, wherein induction ofdifferentiation in the contacted cell identifies the DNA MeTase isoformas a DNA MeTase isoform that is involved in induction of celldifferentiation. In certain preferred embodiments, the agent is anantisense oligonucleotide of the first aspect of the invention. In othercertain preferred embodiments, the agent is an small molecule inhibitorof the first aspect of the invention. In still other certainembodiments, the cell is a neoplastic cell. In still yet other preferredembodiments of the fifth aspect of the invention, the method comprisesan agent of the first aspect of the invention which is a combination ofone or more antisense oligonucleotides and/or one or more small moleculeinhibitors of the first aspect of the invention. In certain preferredembodiments, the DNA MeTase isoform is DNMT-1, DNMT3a, or DNMT3b. Inother certain preferred embodiments, the DNA MeTase isoform is DNMT3aand/or DNMT3b.

[0093] In a sixth aspect, the invention provides a method for inhibitingneoplastic cell growth in an animal comprising administering to ananimal having at least one neoplastic cell present in its body atherapeutically effective amount of an agent of the first aspect of theinvention. In certain embodiments thereof, the agent is an antisenseoligonucleotide, which is combined with a pharmaceutically acceptablecarrier and administered for a therapeutically effective period of time.

[0094] In certain embodiments where the agent of the first aspect of theinvention is a DNA MeTase small molecule inhibitor, therapeuticcompositions of the invention comprising said small moleculeinhibitor(s) are administered systemically at a sufficient dosage toattain a blood level DNA MeTase small molecule inhibitor from about 0.01μM to about 10 μM. In a particularly preferred embodiment, thetherapeutic composition is administered at a sufficient dosage to attaina blood level of DNA MeTase small molecule inhibitor from about 0.05 μMto about 10 μM. In a more preferred embodiment, the blood level of DNAMeTase small molecule inhibitor is from about 0.1 μM to about 5 μM. Forlocalized administration, much lower concentrations than this may beeffective. Preferably, a total dosage of DNA MeTase small moleculeinhibitor will range from about 0.01 mg to about 100 mg protein effectorper kg body weight per day. In a more preferred embodiment, a totaldosage of DNA MeTase small molecule inhibitor will range from about 0.1mg to about 50 mg protein effector per kg body weight per day. In a mostpreferred embodiment, a total dosage of DNA MeTase small moleculeinhibitor will range from about 0.1 mg to about 10 mg protein effectorper kg body weight per day.

[0095] In a seventh aspect, the invention provides a method forinvestigating the role of a particular DNA MeTase isoform in cellularproliferation, including the proliferation of neoplastic cells. In thismethod, the cell type of interest is contacted with an amount of anantisense oligonucleotide that inhibits the expression of one or morespecific DNA MeTase isoforms, as described for the first aspectaccording to the invention, resulting in inhibition of expression of DNAMeTase isoform(s) in the cell. If the contacted cell with inhibitedexpression of the DNA MeTase isoform(s) also shows an inhibition in cellproliferation, then the DNA MeTase isoform(s) is required for theinduction of cell proliferation. In this scenario, if the contacted cellis a neoplastic cell, and the contacted neoplastic cell shows aninhibition of cell proliferation, then the DNA MeTase isoform whoseexpression was inhibited is a DNA MeTase isoform that is required fortumorigenesis. In certain preferred embodiments, the DNA MeTase isoformis DNMT-1, DNMT3a, or DNMT3b. In certain preferred embodiments, the DNAMeTase isoform is DNMT3a and/or DNMT3b.

[0096] Thus, by identifying a particular DNA MeTase isoform that isrequired for in the induction of cell proliferation, only thatparticular DNA MeTase isoform need be targeted with an antisenseoligonucleotide to inhibit cell proliferation or induce differentiation.Consequently, a lower therapeutically effective dose of antisenseoligonucleotide may be able to effectively inhibit cell proliferation.Moreover, undesirable side effects of inhibiting all DNA MeTase isoformsmay be avoided by specifically inhibiting the one (or more) DNA MeTaseisoform(s) required for inducing cell proliferation.

[0097] As previously indicated, the agent of the first aspect includes,but is not limited to, oligonucleotides and small molecule inhibitorsthat inhibit the activity of one or more, but less than all, DNA MeTaseisoforms. The measurement of the enzymatic activity of a DNA MeTaseisoform can be achieved using known methodologies. For example, seeSzyf, M., et al. (1991) J. Biol. Chem. 266:10027-10030.

[0098] Preferably, the DNA MeTase small molecule inhibitor(s) of theinvention that inhibits a DNA MeTase isoform that is required forinduction of cell proliferation is a DNA MeTase small molecule inhibitorthat interacts with and reduces the enzymatic activity of fewer than allDNA MeTase isoforms.

[0099] In an eighth aspect, the invention provides a method foridentifying a DNA MeTase isoform that is involved in induction of celldifferentiation, comprising contacting a cell with an antisenseoligonucleotide that inhibits the expression of a DNA MeTase isoform,wherein induction of differentiation in the contacted cell identifiesthe DNA MeTase isoform as a DNA MeTase isoform that is involved ininduction of cell differentiation. Preferably, the cell is a neoplasticcell. In certain embodiments, the DNA MeTase isoform is DNMT-1, DNMT3a,or DNMT3b. In certain other embodiments, the DNA MeTase isoform isDNMT3a and/or DNMT3b.

[0100] The phrase “inducing cell differentiation” and similar terms areused to denote the ability of a DNA MeTase antisense oligonucleotide orDNA MeTase small molecule inhibitor (or combination thereof) to inducedifferentiation in a contacted cell as compared to a cell that is notcontacted. Thus, a neoplastic cell, when contacted with a DNA MeTaseantisense oligonucleotide or DNA MeTase small molecule inhibitor (orboth) of the invention, may be induced to differentiate, resulting inthe production of a daughter cell that is phylogenetically more advancedthan the contacted cell.

[0101] In a ninth aspect, the invention provides a method for inhibitingcell proliferation in a cell, comprising contacting a cell with at leasttwo of the agents selected from the group consisting of an antisenseoligonucleotide that inhibits a specific DNA MeTase isoform, a DNAMeTase small molecule inhibitor, an antisense oligonucleotide thatinhibits a DNA MeTase, and a DNA MeTase small molecule inhibitor. In oneembodiment, the inhibition of cell growth of the contacted cell isgreater than the inhibition of cell growth of a cell contacted with onlyone of the agents. In certain preferred embodiments, each of the agentsselected from the group is substantially pure. In preferred embodiments,the cell is a neoplastic cell. In yet additional preferred embodiments,the agents selected from the group are operably associated.

[0102] In a tenth aspect, the invention provides a method for modulatingcell proliferation or differentiation comprising contacting a cell withan agent of the first aspect of the invention, wherein one or more, butless than all, DNA MeTase isoforms are inhibited, which results in amodulation of proliferation or differentiation. In preferredembodiments, the cell proliferation is neoplasia. In certainembodiments, the DNA MeTase isoform is selected from DNMT-1, DNMT3a, andDNMT3b. In certain other embodiments, the DNA MeTase isoform is DNMT3aand/or DNMT3b.

[0103] For purposes of this aspect, it is unimportant how the specificDNMT isoform is inhibited. The present invention has provided thediscovery that specific individual DNMTs are involved in cellproliferation or differentiation, whereas others are not. Asdemonstrated in this specification, this is true regardless of how theparticular DNMT isoform(s) is/are inhibited.

[0104] By the term “modulating” proliferation or differentiation ismeant altering by increasing or decreasing the relative amount ofproliferation or differentiation when compared to a control cell notcontacted with an agent of the first aspect of the invention.Preferably, there is an increase or decrease of about 10% to 100%. Morepreferably, there is an increase or decrease of about 25% to 100%. Mostpreferably, there is an increase or decrease of about 50% to 100%. Theterm “about” is used herein to indicate a variance of as much as 20%over or below the stated numerical values.

[0105] The following examples are intended to further illustrate certainpreferred embodiments of the invention and are not limiting in nature.Those skilled in the art will recognize, or be able to ascertain, usingno more than routine experimentation, numerous equivalents to thespecific substances and procedures described herein. Such equivalentsare considered to be within the scope of this invention, and are coveredby the appended claims.

[0106] In an eleventh aspect, the invention provides a method forinhibiting cell proliferation in a cell comprising contacting a cellwith at least two agents selected from the group consisting of anantisense oligonucleotide from the first aspect of the invention thatinhibits expression of a specific DNA MeTase isoform, a small moleculeinhibitor that inhibits a specific DNA MeTase isoform, an antisenseoligonucleotide that inhibits a histone deactylase, and a small moleculethat inhibits a histone deactylase. In one embodiment, the inhibition ofcell growth of the contacted cell is greater than the inhibition of cellgrowth of a cell contacted with only one of the agents. In certainembodiments, each of the agents selected from the group is substantiallypure. In preferred embodiments, the cell is a neoplastic cell. In yetadditional preferred embodiments, the agents selected from the group areoperably associated.

EXAMPLES Example 1 Synthesis and Identification of Active DNMT3a andDNMT3b Antisense Oligonucleotides

[0107] Antisense (AS) were designed to be directed against the 5′- or3′-untranslated region (UTR) of the targeted genes, DNMT3a and DNMT3b.Oligos were synthesized with the phosphorothioate backbone on anautomated synthesizer and purified by preparative reverse-phase HPLC.All oligos used were 20 base pairs in length.

[0108] To identify antisense oligodeoxynucleotide (ODN) capable ofinhibiting DNMT3a or DNMT3b expression in human cancer cells, antisenseoligonucleotides were initially screened in T24 (human blader) A549(human non small cell lung cancers cells at 100 nM. Cells were harvestedafter 24 hours of treatment, and DNMT3a or DNMT3b RNA expression wasanalyzed by Northern blot analysis.

[0109] A total of 27 phosphorothioate ODNs containing sequencescomplementary to the 5′ or 3′ UTR of the human DNMT3a gene (GenBankAccession No. AF067972) were screened as above (FIG. 2). Firstgeneration DNMT3a AS-ODNs with greatest antisense activity to humanDNMT3a were selected for second generation chemistry production. Theseoligonucleotides were then synthesized as second generation chemistry(phosphorothioate backbone and 2′-O-methyl modifications) andappropriate mismatch controls of these were prepared.

[0110] A total of 34 phosphorothioate ODNs containing sequencescomplementary to the 5′ or 3′ UTR of the human DNMT3b gene (GenBankAccession No. NM_(—)006892) were screened as above (FIG. 3). Firstgeneration DNMT3b AS-ODNs with greatest antisense activity to humanDNMT3b were selected for second generation chemistry production. Theseoligonucleotides were then synthesized as second generation chemistry(phosphorothioate backbone and 2′-O-methyl modifications) andappropriate mismatch controls of these were prepared. Table 1 and Table2 provides a summary of oligonucloetides sequences, nucleotide position,and chemical modifications of antisense oligonucleotides targeting theDNMT1, DNMT3a and DNMT3b genes. Sequences of mismatch controloligonucleotides are also given.

Example 2 Dose Dependent Inhibition of DNMT3a and DNMT3b mRNA Expressionwith Antisense Oligonucleotides

[0111] Active oligonucleotides identified in initial screens were thensynthesized with phosporothiate backbone modification and 2′-O-methylmodifications of the sugar on the four 5′ and 3′ nucleotides. In orderto determine whether AS ODN treatment reduced DNMT3a and DNMT3bexpression at the mRNA level dose response experiments were done. HumanA549 or T24 cells were treated with increasing doses of antisense (AS)oligonucleotide from 0-75 nM for 24 hours.

[0112] Briefly, human A549 or T24 human bladder carcinoma cells wereseeded in 10 cm tissue culture dishes one day prior to oligonucleotidetreatment. The cell lines were obtained from the American Type CultureCollection (ATCC) (Manassas, Va.) and were grown under the recommendedculture conditions. Before the addition of the oligonucleotides, cellswere washed with PBS (phosphate buffered saline). Next, lipofectintransfection reagent (GIBCO BRL Mississauga, Ontario, Calif.), at aconcentration of 6.25 μg/ml, was added to serum free OPTIMEM medium(GIBCO BRL, Rockville, Md.), which was then added to the cells. Theoligonucleotides to be screened were then added directly to the cells(i.e., one oligonucleotide per plate of cells).

[0113] Cells were harvested, and total RNAs were analyzed by Northernblot analysis. Briefly, total RNA was extracted using RNeasy miniprepcolumns (QIAGEN). Ten to twenty μg of total RNA was run on aformaldehyde-containing 1% agarose gel with 0.5 M sodium phosphate (pH7.0) as the buffer system. RNAs were then transferred to nitrocellulosemembranes and hybridized with the radiolabelled DNA probes specific forDNMT3a or DNMT3b messenger RNA. Autoradiography was performed usingconventional procedures.

[0114]FIG. 4 presents results of experiments done with a firstgeneration antisense inhibitor of DNMT3a. FIG. 5 is a representativeNorthern blot demonstrating the dose dependent inhibition of DNMT3bexpression by AS-ODN (SEQ ID NO: 18) in A549 human non small cell lungcancer cells (estimated IC₅₀ value of 25 nM). Also demonstrated is thespecificity of SEQ ID NO: 18 for DNMT3b, as non target mRNAs DNMT1,DNMT3A and Glyceraldehyde 3′-phosphate dehydrogenase are not effected.MM indicates control mismatch oligonucleotides.

[0115] Treatment of cells with the indicated AS ODN significantlyinhibits the expression of the targeted mRNA DNMT3a and DNMT3brespectively in a dose dependent fashion in both human A549 and T24cells.

Example 3 DNMT3b Antisense ODNs Inhibit DNMT3b Protein Expression

[0116] In order to determine whether treatment with DNMT3a or DNMT3bAS-ODNs would inhibit expression at the protein level, antibodiesspecific for either DNMT3a or DNMT3b were produced for use in westernblots. DNMT3b is expressed at sufficiently high levels in human cancercells to be detected by our DNMT3b antibody. However, DNMT3a is notexpressed at detectable levels. Therefore, both human A549 non smallcell lung cancer cells and T24 human bladder cancer cells were treatedwith doses of the DNMT3b antisense inhibitor (SEQ ID NO: 18) rangingfrom 0-75 nM for 48 hours and then measured DNMT3b protein levels byWestern blot.

[0117] Briefly, cells were lysed in buffer containing 1% Triton X-100,0.5% sodium deoxycholate, 5 mM EDTA, 25 mM Tris-HC1, pH 7.5, plusprotease inhibitors. Total protein was quantified by the protein assayreagent from Bio-Rad (Hercules, Calif.). 100 ug of total protein wasanalyzed by SDS-PAGE. Next, total protein was transferred onto a PVDFmembrane and probed with DNMT3b specific antibody. Anti-DNMT3b antibodywas raised by immunizing rabbits with a GST fusion protein containing afragment of the DNMT3b protein (amino acids 4-101 of GenBank AccessionNo. NM_(—)006892). Rabbit antiserum was tested and found only to reactspecifically to the human DNMT3b isoform. DNMT3b antiserum was used at1:500 dilution in Western blots to detect DNA MeTase-6 in total celllysates. Horse Radish Peroxidase conjugated secondary antibody was usedat a dilution of 1:5000 to detect primary antibody binding. Thesecondary antibody binding was visualized by use of the Enhancedchemiluminescence (ECL) detection kit (Amersham-Pharmacia Biotech.,Inc., Piscataway, N.J.).

[0118] As shown in FIG. 6, the treatment of T24 or A549 cells withDNMT3b AS-ODN MG3741 inhibits the expression of DNMT3b protein.

Example 4 Effect of DNMT3a and DNMT3b Inhibition on Cancer CellApoptosis and Growth

[0119] In order to determine the effects of DNMT3a and DNMT3b inhibitionon apoptosis of cancer cells, various cancer cell lines (A549 or T24cells, MDAmb231) were exposed to the DNMT3a and DNMT3b AS-ODN forvarious periods of time and the effects on apoptosis were determined.For the analysis of apoptosis (active cell death), cells were analyzedusing the Cell Death Detection ELISA ^(Plus) kit (Roche Diagnostic GmBH,Mannheim, Germany) according to the manufacturer's directions.Typically, 10,000 cells were plated in 96-well tissue culture dishes for2 hours before harvest and lysis. Each sample was analyzed in duplicate.ELISA reading was done using a MR700 plate reader (DYNEX Technology,Ashford, Middlesex, England) at 410 nm. The reference was set at 490 nm.Results of these studies on DNMT3a and DNMT3b inhibition in human cancercells are shown in FIGS. 7-9.

[0120] The effect of DNMT3b inhibition on the induction of apoptosis innormal cells was also determined, the results of which are presented inFIG. 10. HMEC (human mammary epithelial cells, ATCC, Manassas, Va.) andMRHF (male foreskin fibroblasts, ATCC, Manassas, Va.) were treated with75 nM of DNMT3b AS (SEQ ID NO: 18) or its mismatch control SEQ ID NO: 19for 48 hrs as previously described for human cancer cells. FIG. 10 showsthat DNMT3b AS inhibitor does not induce apoptosis in normal cells, butdoes induces apoptosis in cancer cells.

[0121] In order to determine the effects of DNMT3a and DNMT3b inhibitionon the proliferation of cancer cells, various cancer cell lines (A549 orT24 cells, MDAmb231) were exposed to the DNMT3a and DNMT3b AS-ODN forvarious periods of time and the effects on cell proliferation weredetermined. Results of these studies are presented in FIGS. 11A and 11Band demonstrate that the inhibition of DNMT3a or DNMT3b expressiondramatically affects cancer cell proliferation.

[0122] Results of these studies demonstrate that inhibition of DNMT3a orDNMT3b results in growth inhibition and induces apoptosis of humancancer cells but similar inhibition in normal cells does not. As T24cells are p53 null whereas A549 cells have functional p53 protein, theinduction of apoptosis seen is independent of p53 activity. Takentogether these results suggest that inhibition of DNMT3a or DNMT3b mayprovide specific and effective anticancer therapies.

Example 5 Identification of Small Molecule Inhibitors of DNAMethylTransferase Isoforms

[0123] DNA methyltransferase enzymatic activity assays and substratespecificity of the various isoforms are performed as describedpreviously (Szyf, M. et al. (1991) J. Biol. Chem. 266:10027-10030).Briefly, Nuclear extracts are prepared from 1×10⁸ mid-log phase humanH446 cells or mouse Y1 (ATCC, Manassas, Va.) cells which are grown understandard cell culture conditions. Cells are treated with mediumsupplemented with the test compound at a concentration of from about0.001 μM to about 10 mM, or at a concentration of from about 0.01 μM toabout 1 mM, or at a concentration of from about 0.1 μM to about 1 mM.The cells are harvested and washed twice with phosphate buffered saline(PBS), then the cell pellet is resuspended in 0.5 ml Buffer A (10 mMTris pH 8.0, 1.5 mM MgCl₂, 5 mM KCl₂, 0.5 mM DTT, 0.5 mM PMSF and 0.5%Nonidet P40) to separate the nuclei from other cell components. Thenuclei are pelleted by centrifugation in an Eppendorf microfuge at 2,000RPM for 15 min at 4° C. The nuclei are washed once in Buffer A andre-pelleted, then resuspended in 0.5 ml Buffer B (20 mM Tris pH 8.0,0.25% glycerol, 1.5 mM MgCl₂, 0.5 mM PMSF, 0.2 mM EDTA 0.5 mM DTT and0.4 mM NaCl). The resuspended nuclei are incubated on ice for 15 minutesthen spun at 15,000 RPM to pellet nuclear debris. The nuclear extract inthe supernatant is separated from the pellet and used for assays for DNAMeTase activity.

[0124] For each assay, carried out in triplicate, 3 μg of nuclearextract is used in a reaction mixture containing 0.1 μg of a synthetic33-base pair hemimethylated DNA molecule substrate with 0.5 μCiS-[methyl-³ H] adenosyl-L-methionine (78.9 Ci/mmol) as the methyl donorin a buffer containing 20 mM Tris-HCl (pH 7.4), 10 mM EDTA, 25%glycerol, 0.2 mM PMSF, and 20 mM 2-mercaptoethanol. The reaction mixtureis incubated for 1 hour at 37° C. to measure the initial rate of the DNAMeTase activity. The reaction is stopped by adding 10% TCA toprecipitate the DNA, then the samples are incubated at 4° C. for 1 hourand the TCA precipitates are washed through GFC filters (Fischer,Hampton, N.H.). Controls are DNA incubated in the reaction mixture inthe absence of nuclear extract, and nuclear extract incubated in thereaction mixture in the absence of DNA.

[0125] The filters are laid in scintillation vials containing 5 ml ofscintillation cocktail, and tritiated methyl groups incorporated intothe DNA are counted in a scintillation counter according to standardmethods. To measure inhibition of DNA MeTase expression, the specificactivity of the nuclear extract from test compound-treated cells iscompared with the specific activity of the extract from untreated cells.Treatment of cells with test compounds that are candidate small moleculeinhibitors of DNA MeTase activity will result in a reduction in DNAMeTase activity in the nuclear extract.

[0126] The above assay may be easily adapted for testing the affect oftest compounds on the activity of individual, recombinantly produced,DNA MeTase isoforms. In order to produce recombinant protein for eachDNA MeTase isoform, an expression construct was produced for eachisotype (Dnmt1, Dnmt3a and Dnmt3b (Dnmt3b2 and Dnmt3b3 splice variants))by inserting the entire coding sequence of the respective isotype intothe pBlueBac4.5™ baculovirus expression vector(Invitrogen, Carlsbad,Calif.). Each construct was then used to infect High Five insect cellsaccording to Invitrogen's baculovirus expression manual.

[0127] Purification of baculovirus expressed human Dnmt1, Dnmt3a andDnmt3b proteins was done as follows: Nuclear extract was isolated fromHigh Five insect cells, the salt concentration was adjusted with buffer(20 mM Tris pH 7.4, 1 mM Na₂EDTA, 10% sucrose) to get a finalconcentration of 0.1M NaCl. Lysate was centrifuged at 9500 g for 10 min.Supernatant was applied to Q-sepharose, Heparin, and Source Q15 columnsequentially. All purifications are performed on a gradifrac system witha P1 pump at 4° C.

[0128] DNA MeTase isotype specific activity assays are performedaccording to the following procedure. From about 100 pg to about 25 μg,or more preferably from about 10 ng to about 10 μg, or most preferablyfrom about 100 ng to about 2.5 μg of recombinant DNA MeTase isotypeprotein is incubated in a reaction mixture containing 0.1 μg of asynthetic 33-base pair hemimethylated DNA molecule substrate with 0.5μCi S-[methyl-³ H] adenosyl-L-methionine (78.9 Ci/mmol) as the methyldonor in a buffer containing 20 mM Tris-HCl (pH 7.4), 10 mM EDTA, 25%glycerol, 0.2 mM PMSF, and 20 mM 2-mercaptoethanol in a total volume of30 μl. Test sample also includes the test small molecule inhibitorcompound at a concentration of from about 0.001 μM to about 10 mM, or ata concentration of from about 0.01 μM to about 1 mM, or at aconcentration of from about 0.1 μM to about 1 mM. The reactions arestopped and the samples are processed as described herein above.

[0129] It is expected that certain candidate small molecule inhibitorsof DNA MeTase activity will have the affect of significantly decreasingthe amount of radioactive methyl incorporated into the substrate DNA.

EQUIVALENTS

[0130] Those skilled in the art will recognize, or be able to ascertain,using no more than routine experimentation, many equivalents to thespecific embodiments of the invention described herein. Such equivalentsare intended to be encompassed by the following claims.

What is claimed is
 1. An agent that inhibits one or more specific DNAmethyltransferase isoforms, but less than all DNA methyltransferaseisoforms, wherein the agent is selected from the group consisting of ananti-DNA methyltransferase oligonucleotide and a small moleculeinhibitor of DNA methyltransferase.
 2. The agent according to claim 1that is an oligonucleotide.
 3. The oligonucleotide according to claim 2,wherein the oligonucleotide is a chimeric oligonucleotide.
 4. Theoligonucleotide according to claim 2, wherein the oligonucleotide is ahybrid oligonucleotide.
 5. The oligonucleotide according to claim 2,wherein the oligonucleotide is complementary to a region of RNA ordouble-stranded DNA selected from the group consisting of (a) a nucleicacid molecule encoding at least 13 contiguous oligonucleotides fromDNMT-1 (SEQ ID NO: 1), (b) a nucleic acid molecule encoding at least 13contiguous oligonucleotides from DNMT3a (SEQ ID NO: 2), and (c) anucleic acid molecule encoding at least 13 contiguous oligonucleotidesfrom DNMT3b (SEQ ID NO: 3).
 6. The oligonucleotide according to claim 5having a nucleotide sequence of from about 13 to about 35 nucleotides.7. The oligonucleotide according to claim 5 having a nucleotide sequenceof from about 15 to about 26 nucleotides.
 8. The oligonucleotideaccording to claim 5 having one or more phosphorothioate internucleosidelinkage, being 20-26 nucleotides in length, and being modified such thatthe terminal four nucleotides at the 5′ end of the oligonucleotide andthe terminal four nucleotides at the 3′ end of the oligonucleotide eachhave 2′-O-methyl groups attached to their sugar residues.
 9. Theoligonucleotide according to claim 5, wherein the oligonucleotide iscomplementary to a region of RNA or double-stranded DNA encoding aportion of DNMT1 (SEQ ID NO: 1).
 10. The oligonucleotide according toclaim 5 that is selected from the group consisting of SEQ ID NO: 4, SEQID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, andSEQ ID NO:
 10. 11. The oligonucleotide according to claim 5, wherein theoligonucleotide is complementary to a region of RNA or double-strandedDNA encoding a portion of DNMT3a (SEQ ID NO: 1).
 12. The oligonucleotideaccording to claim 11 that is selected from the group consisting of SEQID NO: 28, SEQ ID NO: 29, SEQ ID NO: 30, SEQ ID NO: 31, SEQ ID NO: 33,SEQ ID NO: 34, and SEQ ID NO:
 36. 13. The oligonucleotide according toclaim 5, wherein the oligonucleotide is complementary to a region of RNAor double stranded DNA encoding a portion of DNMT3b (SEQ ID NO: 3). 14.The oligonucleotide according to claim 13 that is selected from thegroup consisting of SEQ ID NO: 18, SEQ ID NO: 20, SEQ ID NO: 21, SEQ IDNO: 22, SEQ ID NO: 23, SEQ ID NO: 25, SEQ ID NO: 26, and SEQ ID NO: 27.15. A method for inhibiting one or more DNA methyltransferase isoformsin a cell comprising contacting the cell with the agent according toclaim
 1. 16. A method for inhibiting one or more DNA methyltransferaseisoforms in a cell comprising contacting the cell with theoligonucleotide according to claim
 2. 17. The method according to claim16, wherein cell proliferation is inhibited in the contacted cell. 18.The method according to claim 16, wherein the oligonucleotide thatinhibits cell proliferation in a contacted cell induces the contactedcell to undergo growth retardation.
 19. The method according to claim16, wherein the oligonucleotide that inhibits cell proliferation in acontacted cell induces the contacted cell to undergo growth arrest. 20.The method according to claim 16, wherein the oligonucleotide thatinhibits cell proliferation in a contacted cell induces the contactedcell to undergo programmed cell death.
 21. The method according to claim16, wherein the oligonucleotide that inhibits cell proliferation in acontacted cell induces the contacted cell to undergo necrotic celldeath.
 22. The method according to claim 16, further comprisingcontacting the cell with a DNA methyltransferase small moleculeinhibitor.
 23. A method for inhibiting neoplastic cell proliferation inan animal comprising administering to an animal having at least oneneoplastic cell present in its body a therapeutically effective amountof the agent of claim
 1. 24. A method for inhibiting neoplastic cellproliferation in an animal comprising administering to an animal havingat least one neoplastic cell present in its body a therapeuticallyeffective amount of the oligonucleotide of claim
 2. 25. The methodaccording to claim 24, wherein the animal is a human.
 26. The methodaccording to claim 24, further comprising administering to the animal atherapeutically effective amount of a DNA methyltransferase smallmolecule inhibitor with a pharmaceutically acceptable carrier for atherapeutically effective period of time.
 27. The method according toclaim 26, wherein the animal is a human.
 28. A method for identifying aDNA methyltransferase isoform that is required for the induction of cellproliferation, the method comprising contacting the DNAmethyltransferase isoform with an inhibitory agent, wherein a decreasein the induction of cell proliferation indicates that the DNAmethyltransferase isoform is required for the induction of cellproliferation.
 29. The method according to claim 28, wherein theinhibitory agent is an oligonucleotide of claim
 2. 30. A method foridentifying a DNA methyltransferase isoform that is required for cellproliferation, the method comprising contacting the DNAmethyltransferase isoform with an inhibitory agent, wherein a decreasein cell proliferation indicates that the DNA methyltransferase isoformis required for cell proliferation.
 31. The method according to claim30, wherein the inhibitory agent is an oligonucleotide of claim
 2. 32. Amethod for identifying a DNA methyltransferase isoform that is requiredfor the induction of cell differentiation, the method comprisingcontacting the DNA methyltransferase isoform with an inhibitory agent,wherein an induction of cell differentiation indicates that the DNAmethyltransferase isoform is required for the induction of cellproliferation.
 33. The method according to claim 32, wherein theinhibitory agent is an oligonucleotide of claim
 2. 34. A method forinhibiting cell proliferation in a cell, comprising contacting a cellwith at least two agents selected from the group consisting of anantisense oligonucleotide that inhibits a specific DNA methyltransferaseisoform, a DNA methyltransferase small molecule inhibitor that inhibitsa specific DNA methyltransferase isoform, an antisense oligonucleotidethat inhibits a DNA methyltransferase, and a DNA methyltransferase smallmolecule inhibitor.
 35. A method for modulating cell proliferation ordifferentiation of a cell comprising inhibiting a specific DNAmethyltransferase isoform that is involved in cell proliferation ordifferentiation by contacting the cell with an agent of claim
 1. 36. Themethod according to claim 35, wherein the cell proliferation isneoplasia.
 37. The method according to claim 36, wherein the DNAmethyltransferase isoform is selected from the group consisting ofDNMT-1, DNMT3a and DNMT3b.
 38. The method according to claim 37, whereinthe DNA methyltransferase isoform is selected from DNMT3a and DNMT3b.39. The method according to claim 37, wherein the DNA methyltransferaseis DNMT3b.